Project Summary/Abstract Carbon monoxide (CO) poisoning remains a major cause of death and disability, affecting 50,000 people per year in the United States alone. Patients removed from fires or following exposure to car and home generator exhaust are placed on 100% oxygen and transferred to a facility with a hyperbaric oxygen delivery system. Despite the availability of hyberbaric therapy centers in most major cities, inherent delays in access to and initiation of therapy greatly limit efficacy. In fact, even with hyberbaric oxygen therapy, 1-2% of patients die and >25% of surviving patients exhibit neurocognitive impairments. There is currently no point-of-care antidote for CO poisoning available clinically. In the present proposal, we continue our studies developing novel antidotal therapies for CO poisoning, based on our findings that extremely high affinity heme-based molecules can bind and sequester CO from red blood cells and tissue mitochondria to reverse the systemic ischemia of CO poisoning. In the previous funding period, we discovered a surprising and near-irreversible CO-binding affinity of mutationally engineered human neuroglobin (Ngb). Ngb is a six-coordinate hemoprotein, with the heme iron coordinated by two histidine residues. We mutated the distal histidine to glutamine (H64Q) and three surface-thiols to form a five-coordinate heme protein (Ngb-H64Q-CCC) that has very high solubility (>10mM), allowing for high concentration and intravenous infusion. This molecule binds CO ~ 500 times more strongly than hemoglobin. Infusions of Ngb- H64Q-CCC in CO-poisoned mice enhanced CO removal from red blood cells in vivo from 25 minutes to 25 seconds, restored heart rate and blood pressure, increased survival from less than 10% to over 85%, and were followed by rapid renal elimination of CO-bound Ngb-H64Q-CCC. These findings provide proof-of-concept that heme-based scavenger molecules with very high CO binding affinity can be developed as potential antidotes for CO poisoning. We aim to continue development of our Ngb-H64Q-CCC molecule, evaluating efficacy on the restoration of cellular aerobic respiration, safety, and acute- and long-term effects on cardiovascular and cognitive function and survival in pre-clinical models, and scaling production of recombinant protein for clinical development. We also aim to further discover novel CO scavenger small molecules, based on knowledge derived from our previous studies. We will mutationally engineer heme-bound, small 6-12 amino acid peptides derived from microperoxidase, with and without iron-to- cobalt metal substitutions to limit redox reactivity and enhance CO affinity. Overall, these proposed studies are in keeping with the mission of the NHLBI and NIH to advance highly impactful, significant, and novel studies that have great potential to improve the public health. Support for these proposed studies has the potential to change our current paradigm for therapy of CO poisoning.